Globular Proteins I PDF

MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo GLOBULAR PROTEINS I Describe the folding mechanism of a globular protein Protein folding is a highly ordered process by which a linear polypeptide chain acquires its functional 3D...

MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo GLOBULAR PROTEINS I Describe the folding mechanism of a globular protein Protein folding is a highly ordered process by which a linear polypeptide chain acquires its functional 3D structure. The process involves 7 stages, each contributing to a final, stable conformation. Stages of Protein Folding 1. Translation: o The ribosome synthesizes the polypeptide chain by linking amino acids in the sequence dictated by mRNA. o The chain starts as an unfolded, linear structure. 2. Secondary Structure Formation: o Local regions of the protein adopt repetitive structural motifs - alpha-helices & beta-sheets through hydrogen bonding between backbone atoms. o These structures provide the initial framework for folding. 3. Hydrophobic Collapse: o Hydrophobic amino acid side chains aggregate & bury themselves inside the protein, minimizing their exposure to H2O. o This creates a loosely packed hydrophobic core & initiates the formation of a compact intermediate, often referred to as the molten globule state. 86 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo 4. Water Exclusion and Core Formation: o Water molecules are excluded from the hydrophobic interior as the protein folds more tightly. o Excluding H20 stabilizes the folding process & minimizes the energy cost of exposing hydrophobic residues to aqueous environment. 5. Domain Interactions: o If the protein has distinct structural domains (independent folding units), these domains interact & position themselves relative to one another. o Interactions between domains help guide the formation of the functional 30 structure. 6. Internal Packing: o The protein achieves its final 30 structure by tightly packing side chains in the hydrophobic core & optimising weak interactions such as: § Hydrogen bonds § Van der Waals forces § Ionic interactions o This stage locks the protein into its most thermodynamically stable configuration. 7. Quaternary Structure Assembly (if applicable): o For proteins with multiple polypeptide chains (subunits), these subunits assemble into a functional multimeric complex. o This assembly involves interactions such as hydrophobic contacts, hydrogen bonding, & ionic interactions. 87 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Key Considerations in Folding Cofactor Interactions: o Some proteins require cofactors or prosthetic groups (permanetly associated co-facotors) to achieve their final functional structure. o Haemoglobin requires a heme group for O2 binding & enzymes may need metal ions (zinc or magnesium) or organic molecules like FAD or NAD+ to function properly. Phi/Psi Angles: o The folding process adheres to sterically allowed regions of Ramachandran plots, which predicts favorable backbone conformations for amino acid residues. o Glycine is highly flexible due to its small size & lack of a side chain; enabling it to occupy regions on the Ramachadran plots that are dissallowed for most other amino acids. o Proline has a rigid, cyclic structure, that restricts its backbone flexibility. This makes it a helix breaker in alpha-helices & limits its conformational role in 20 structures. Chaperones: o Molecular chaperones assist during folding by preventing aggregation & ensuring correct structure formation. Folding Challenges: o Protein misfolding can result in aggregation & the formation of insoluble structures, leading to diseases known as amyloidoses. o These diseases include: Alzheimer’s disease (amyloid-beta peptides & tau proteins), Parkinson’s disease (alpha-synuclein into Lewy bodies), & Creutzfeldt-Jakob disease (prion aggregation). 88 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Explain how molecular chaperones aid proteins Molecular chaperones & their role in protein folding Chaperones, also known as heat shock proteins (HSPs), are specialized proteins (produced in response to heat stress) that assist other proteins in achieving their correct conformations. They help at various points in the folding process: o Prevent premature folding during synthesis by stabilizing unfolded or partially folded intermediates. o Catalyze folding process, ensuring efficient transition to native state. o Protect vulnerable regions of a protein from forming incorrect interactions or aggregates. Proteins and their dependency on chaperones: o Some proteins completely require chaperones to fold correctly; without assistance, they cannot achieve their functional conformations. o However, most proteins can fold independently, although chaperones like Hsp70 can enhance efficiency. 89 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Types of Molecular Chaperones Hsp70 Family § Plays a significant role in ensuring proper protein folding during translation. § Binds to hydrophobic residues of nascent proteins as they exit the ribosome; § Prevents premature folding or aggregation of polypeptide chain. § Does not require ATP for its action; unlike other chaperones. Hsp60/10 (GroEL/ES in bacteria) § Found in mitochondria; critical for protein folding within this organelle. § Forms a specialised folding chamber that isolates individual proteins; allowing them to fold correctly without interference. § Essential for life; deletion leads to organismal non-viability. Hsp90 Family: § A large, complex chaperone that assists in folding a wide range of proteins. § Binds to specific co-chaperones depending on its client protein, aiding their folding. § Facilitates the folding of aberrant proteins, potentially contributing to diseases such as tumorigenesis or neurodegenerative diseases. § Clinical Relevance: target of interest for drug development, due to its role in supporting cancer progression. 90 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Chaperones in the ER lumen Binding Protein (BiP): o Member of the Hsp70 family. o Interacts with nascent proteins as they enter the ER lumen o Prevents aggregation & ensures proper folding of newly synthesized proteins Calnexin: o A specialized chaperone that also binds calcium ions. o Interacts with carbohydrate modifications on proteins (PTMs) to ensure proper folding. o Known to retain misfolded proteins in ER for quality control. Chaperone for Haemoglobin Alpha Haemoglobin Stabilizing Protein (AHSP): o Specifically stabilizes alpha-globin chains in the absence of beta- globin chains during haemoglobin assembly. o Prevents the accumulation and aggregation of free alpha-globin chains, which could otherwise lead to toxicity. Significance of AHSP o In the absence of beta chains, alpha chains may form Hb Barts (α4), which is non-function al & associated with severe anaemia. 91 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Describe the Ubiquitin-proteasomase system (UPS) in brief The UPS is a cellular mechanism for targeted protein degradation, critical for maintaining protein homeostasis and regulating various biological processes. 92 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Ubiquitination Proteins destined for degradation are tagged with a polyubiquitin chain, requiring at least 4 ubiquitin molecules. The process is carried out by 3 types of enzymes: o E1 (Ubiquitin-activating enzyme): Activates ubiquitin in an ATP- dependent manner. o E2 (Ubiquitin-conjugating enzyme): Transfers ubiquitin from E1 to the target protein. o E3 (Ubiquitin ligase): The key enzyme that identifies and binds the target protein, ensuring specificity. Over 600 E3 ligase genes exist in the human genome. Proteasome Recognition & Degradation Polyubiquitinated proteins are recognized by the alpha subunits of the 26S proteasome. Beta subunits of the proteasome carry out proteolysis, breaking down the protein into peptide fragments. Ubiquitin is recycled for reuse. Structure of the 26S Proteasome Composed of a 20S core particle flanked by 19S regulatory particles. 20S core: o 7 di]erent alpha subunits recognize ubiquitinated proteins. o 7 di]erent beta subunits possess proteolytic activity with varying substrate specificity. 93 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Explain what amyloidosis is Amyloidosis refers to a group of disorders characterized by the abnormal deposition of insoluble amyloid fibrils in tissues and organs. These fibrils are formed from misfolded proteins that aggregate into β-pleated sheet structures stabilised by H-bonding, leading to damage & dysfunction. Amyloid Formation Amyloid as a natural folding state o Amyloid formation may represent a potential folding state for all proteins (an intermediate folding step). o Under normal conditions, protein chaperones continuously work to prevent proteins from adopting this conformation. o All amyloid-forming proteins are normal & essential to the body. Stages of Amyloid Formation o Misfolding: occurs when hydrophobic fragments of a protein, usually buried within its core, are exposed to the aqueous environment. o The intermediate misfolded state has a high tendency to aggregate. o Oligomerisation & Fibril formation: misfolded protein intermediates forms oligomeric β-sheet structures, which further grow into protofibrils & finally become cross β-amyloid like-fibrils. o Stabilisation: amyloid fibrils are stabilized by hydrogen bonding in their B-sheet conformations, & the side-chains of proteins are not vital for this process. o Even proteins with highly diverse sequences can form amyloids. 94 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Impact of Amyloids on Organs Brain o Amyloid accumulation in the brain leads to noticeable & early symptoms due to brain’s sensitivity & complexity. o Even a small amount of amyloid buildup in the brain can cause significant neurological damage. Other organs o Larger organs such as the liver or heart, can tolerate amyloid accumulation for extended periods before symptoms emerge. o Substantial amyloid deposits may remain unnoticed until significant damages occur. Delayed Onset of Symptoms o Amyloid buildup & the associated clinical symptoms often have a long latency period. o Studies suggest (in the case of a variant of Creutzfeldt-Jakob disease) that it may take up to 35 years for amyloid accumulation to reach levels that impact health. o Sometimes they do not cause disease & surgeons have discovered them by chance when operating for another unrelated condition. 95 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Amyloidosis (Amyloid Diseases) ALZHEIMER’S DISEASE Amyloid β (Aβ) & Plaque Formation: o The dominant component of amyloid plaques in Alzheimer’s disease (AD) is amyloid β (Aβ), a peptide consisting of 40–42 amino acids. o Structural Features: X-ray crystallography & infrared spectroscopy reveal a characteristic β-pleated sheet conformation in non-branching fibrils. o Neurotoxicity: Aβ peptides aggregate in a β-pleated sheet configuration, forming amyloid plaques that are neurotoxic. This aggregation is considered a central pathogenic event, contributing to cognitive impairment in AD. Amyloid Precursor Protein (APP) o Aβ is derived from proteolytic cleavage of the APP, a transmembrane protein expressed on the cell surface of neurons & other tissues. o APP plays a role in neural stem cell development & neurorepair under normal conditions. o The cleaved Aβ peptides aggregate to form amyloid plaques in the brain parenchyma & around blood vessels. 96 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Genetic & Sporadic Cases o The majority of Alzheimer’s disease are sporadic (not inherited). However, 5-10% of cases are familial, linked to genetic mutations in APP, presenilin 1, or presenilin 2 genes. Neurofibrillary Tangles & Tau Protein o A second key factor in Alzheimer’s pathology is the accumulation of the neurofibrillary tangles inside neurons. o These tangles consist of an abnormal form of tau (t) protein. In its normal form, tau supports microtubule assembly, which is critical for intracellular transport. o Abnormal tau blocks the activity of its functional counterpart, disrupting microtubule structure & further contributing to neuronal dysfunction & eventually death. Implications o Both extracellular Aβ plaques & intracellular tau tangles are a hallmark features of Alzheimer’s disease. o Research continue to explore how Aβ aggregation & tau dysfunction interact to drive the progression of AD. 97 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo PARKINSON’S DISEASE Protein Involved: Alpha-synuclein Mechanism: o α-synuclein misfolds & aggregatese into Lewy bodies, toxic structures that accumulate in neurons. o This disrupts neuronal function; contributing to motor & cognitive symtpoms. Creutzfeldt-Jakob Disease (CJD) Protein Involved: Prion protein (PrP) Mechanism: o A normal prion protein undergoes a conformational change into an abnormal misfolded form (known as the scrapie infectious form; named after prion disease in sheep) which induces other PrP to misfold. o This eventually leads to the formation of amyloid fibrils; which aggregate & deposit in the brain tissue. o Over time, these aggregates cause transmissible spongiform encephalopathy (TSE) – characterised by sponge-like holes in brain tissue. Type 2 Diabetes Mellitus Protein Involved: Amylin (Islet Amyloid Polypeptide, IAPP) Mechanism: o Amylin aggregates into amyloid deposits in the pancreatic islets; disrupting insulin secretion & contributing to beta-cell death. o NOTE: Amylin is in many ways similar to C-peptide in that it is a polypeptide hormone secreted at the same time as insulin (much lower amounts though 1/100). 98 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Fatal Familial Insomnia (FFI) Protein Involved: Prion protein (PrP) Mechanism: o Similar to CJD, with prion aggregates in specific brain region (e.g. thalamus), leading to severe sleep disruption & neurodegeneration. o The mutation in the PRNP gene is associated with this condition & alters the folding of the PrP protein, driving the disease progression. Primary Amyloidosis (AL Amyloidosis) Protein Involved: Immunoglobulin light chains Mechanism: o Misfolded light chains from plasma cells form amyloid fibrils; depositing in tissues such as the heart, kidney & liver. o This condition is often associated with plasma cell dyscrasias, which drives the overproduction of monoclonal light chains. Hereditary Transthyretin Amyloidosis (ATTR) Protein Involved: Transthyretin (TTR) Mechanism: o Mutations destabilise the TTR tetramer, causing monomers to misfold & aggregate into amyloid fibrils. o Fibril deposition commonly affects the heart (left ventricle) leading to cardiomyopathy & may involve peripheral nerves. o Normal TTR Structure: tetramer synthesized in the liver, with 2 thyroxine-binding sites per tetramer. o Normal TTR Function: transports thyroxine (T4) & vitamin A in the blood 99 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Describe how prion proteins are transmissible Prion proteins are unique infectious agents that propagate by inducing a conformational change in their normal counterparts. This mechanism underlies the transmissibility of prion diseases, such as Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (mad cow disease) in cattle. 100 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Normal Prion Protein (PrPc) Structure PrPC is a normal cellular protein present on the surface of neurons & glial cells. It is involved in various cellular functions (e.g. cell signalling). It is encoded by the host's genome & typically adopts an alpha-helical structure. PrPC is not inherently infectious & is susceptible to proteolytic degradation. Infection Prion Protein (PrPSc) Structure PrPSc is a misfolded version of PrPC; characterised by a beta-sheet-rich conformation. This structural change makes PrPSc highly resistant to proteolytic digestion & prone to forming insoluble aggregates. High Energy Barrier for Conversion Under normal conditions, the conversion of PrPC to PrPSc is prevented by a high energy activation barrier; making spontaneous misfolding extremely rare. Triggering Misfolding by Exogenous PrPSc Structure When PrPSc is introduced into the body (e.g. through ingestion or inoculation), it interacts with endogenous PrPC. PrPSc acts as a “template,” promoting the rapid refolding of PrPC into the infectious beta-sheet-rich conformation. Exponential Propogation Newly converted PrPSc molecules trigger additional PrPC to misfold; resulting in exponential propagation of infectious prions. Aggregates of PrPSc form multimeric assemblies; further stabilising the pathogenic state & promoting more conversions. 101 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo Proteolytic Resistance & Aggregation PrPSc aggregates into amyloid-like fibrils resistant to proteolytic digestion. These aggregates accumulate in brain tissue, forming hallmark lesions of TSEs. Disease Pathology The accumulation of PrPSc aggregates disrupts normal brain function, leading to neuronal degeneration & characteristics sponge-like holes in brain tissue. This process ultimately results in neurodegeneration, severe symptoms, & invariably fatal outcomes. 102